Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local alpha-helical form is present around Gly10 whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local beta-sheet form as viewed from Gly10 and Phe22, and both beta-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from alpha-helix to beta-sheet, and from random coil to beta-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local beta-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic alpha-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel beta-sheets at pH 7.5 and a mixture of antiparallel and parallel beta-sheets at pH 3.3.     

Kinetics of denaturation of human and chicken hemoglobins in the presence of co-solvents

The stability of four hemoglobins (Hb) in dimer forms (low concentration) were investigated by the kinetics of denaturation. The rate constants of denaturation were obtained by variation of 280 nm absorption versus time in 10 mM Tris-HCl, 10 mM EDTA, pH 8.0 at 45 degrees C in the absence and presence of 0.5 M ethanol, dimethyl sulfoxide (DMSO), formamide, and glycerol. The results show the trend of rate constants in different co-solvents in the following order: chicken hemolysate < human hemolysate and chicken Hb D < chicken Hb A. The buried surface area was calculated for Hb samples in the absence of co-solvents. Accordingly, the trend points out that: chicken Hb D > chicken Hb A > human Hb A. These results suggest that both chicken hemolysate and chicken Hb D are relatively more stable than human and chicken Hb A, respectively. However, the denaturation rate constants of Hb in different co-solvents have designated the following order: ethanol > DMSO > formamide > glycerol. As a matter of fact, this phenomenon is an indication of an increase in the denaturation capacity (DC) and hydrophobicity, and a decrease in the surface tension of the solution in the preceding co-solvents.     

Structural dynamic of a self-assembling peptide d-EAK16 made of only D-amino acids

We here report systematic study of structural dynamics of a 16-residue self-assembling peptide d-EAK16 made of only D-amino acids. We compare these results with its chiral counterpart L-form, l-EAK16. Circular dichroism was used to follow the structural dynamics under various temperature and pH conditions. At 25 degrees C the d-EAK16 peptide displayed a typical beta-sheet spectrum. Upon increasing the temperature above 70 degrees C, there was a spectrum shift as the 218 nm valley widens toward 210 nm. Above 80 degrees C, the d-EAK16 peptide transformed into a typical alpha-helix CD spectrum without going through a detectable random-coil intermediate. When increasing the temperature from 4 degrees C to 110 degrees C then cooling back from 110 degrees C to 4 degrees C, there was a hysteresis: the secondary structure from beta-sheet to alpha-helix and then from alpha-helix to beta-sheet occurred. d-EAK16 formed an alpha-helical conformation at pH0.76 and pH12 but formed a beta-sheet at neutral pH. The effects of various pH conditions, ionic strength and denaturing agents were also noted. Since D-form peptides are resistant to natural enzyme degradation, such drastic structural changes may be exploited for fabricating molecular sensors to detect minute environmental changes. This provides insight into the behaviors of self-assembling peptides made of D-amino acids and points the way to designing new peptide materials for biomedical engineering and nanobiotechnology.     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.     

The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.     

Conformational study of globulin from rice (Oryza sativa) seeds by Fourier-transform infrared spectroscopy

The conformation of rice globulin (10%, w/v, in deuterated phosphate buffer, pD 7.4) under the influence of pH, chaotropic salts, several protein structure perturbants and heat treatments was studied by Fourier-transform infrared (FTIR) spectroscopy. Rice globulin exhibited seven major bands in the region of 1700-1600 cm-1 and the spectrum suggests high alpha-helical content with large quantities of beta-sheet and beta-turn structures. Highly acidic and alkaline pH conditions induced changes in band intensity attributed to intermolecular beta-sheet structure (1681 and 1619 cm-1). Addition of chaotropic salts led to progressive changes in band intensity, following the lyotropic series of anions, whereas several protein structure perturbants caused shifts in band positions. Heating at increasing temperature led to progressive decreases in alpha-helical content and increases in random coil structures, suggesting protein denaturation. This was accompanied by intensity increases in the intermolecular beta-sheet transitions.     

Intermediacy of poly(L-proline) II and beta-strand conformations in poly(L-lysine) beta-sheet formation probed by temperature-jump/UV resonance Raman spectroscopy

Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperature jump (T-jump) was used to investigate early steps in the temperature-induced alpha-helix to beta-sheet conformational transition of poly(L-lysine) [poly(K)]. Excitation at 197 nm from a tunable frequency-quadrupled Ti:sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although un-ionized poly(K) (pH 11.6) is mainly alpha-helical below 30 degrees C, there is a detectable fraction (approximately 15%) of unfolded polypeptide, which is mainly in the poly(L-proline) II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be beta-strand. Above 30 degrees C un-ionized poly(K) forms a beta-sheet at a rate (minutes) which increases with increasing temperature. A 22-44 degrees C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/beta-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, approximately 2% at the 3 micros time limit of the experiment. The rise time of the PPII signals is approximately 250 ns, consistent with melting of short alpha-helical segments. A model is proposed in which the melted PPII segments interconvert with beta-strand conformation, whose association through interstrand H-bonding nucleates the formation of beta-sheet. The intrinsic propensity for beta-strand formation could be a determinant of beta-sheet induction time, with implications for the onset of amyloid diseases.     

Conformational stability of 17 beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus

The functional activities of proteins are closely related to their molecular structure and understanding their structure-function relationships remains one of the intriguing problems of molecular biology. We investigated structural changes in 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) induced by pH, temperature, salt, urea, guanidine hydrochloride, and coenzyme NADPH binding. At 25 degrees C and within the relatively narrow pH range of 7.0-9.0, 17beta-HSDcl exists in its native conformation as a dimer. This native conformation is thermally stable up to 40 degrees C in this pH range. At 25 degrees C and pH 2.0 in the presence of 150-300 mM NaCl, 17beta-HSDcl forms soluble aggregates enriched in alpha-helical and beta-sheet structures. At higher temperatures and NaCl concentrations, these soluble aggregates start to precipitate. The denaturants urea and guanidine hydrochloride unfold 17beta-HSDcl at concentrations of 1.2 and 0.4 M, respectively. Binding of the coenzyme NADPH to 17beta-HSDcl causes local structural changes that do not significantly affect the thermal stability of this protein.     

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.     

Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm

The objective of the present study was to investigate the synergistic effect of DMSO and glycerol added at various temperatures on the post-thaw quality of buffalo sperm. Pooled ejaculates from four Nili-Ravi buffalo bulls were divided into 18 aliquots and extended (1:10) in Tris-citric acid extender differing in glycerol:DMSO ratios (0:0, 0:1.5, 0:3; 3:0, 3:1.5, 3:3; and 6:0, 6:1.5, 6:3, respectively; %, v:v) either at 37 or 4 degrees C. Semen was packaged in 0.5 mL French straws and frozen in a programmable cell freezer. Thawing was performed at 37 degrees C for 50s. Post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo sperm were determined using computer-assisted semen analyzer (CASA), hypoosmotic swelling (HOS) assay and phase-contrast microscopy, respectively. Glycerol (6%) in extender yielded better post-thaw sperm motility, velocities (straight-line and average path), plasma membrane integrity, and normal acrosomes (P<0.05). Post-thaw sperm motility and plasma membrane integrity declined in the presence of DMSO (P<0.01). The addition of glycerol (6%) at 37 degrees C yielded better post-thaw sperm motility, plasma membrane integrity and velocities than addition at 4 degrees C (P<0.05). In conclusion, glycerol is still an essential cryoprotectant for buffalo sperm. The addition of DMSO antagonized the cryoprotection ability of glycerol and reduced the post-thaw quality of buffalo sperm. Furthermore, 6% glycerol added at 37 degrees C, provided better cryoprotection to the motility apparatus and plasma membrane integrity of buffalo sperm.     

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.     

Laser Raman characterization of conformational changes in sarcoplasmic reticulum induced by temperature, Ca2+, and Mg2+

Laser Raman spectroscopy has been used to examine the conformations of the protein and phospholipid components of sarcoplasmic reticulum from rabbit white skeletal muscle. The phospholipid component is shown to have the conformation of fluid, liquid-crystalline lipids, even at 10 degrees C, and no breaks in the lipid conformation are observed in the range of 10-37 degrees C. Protein (predominantly the Ca2+-dependent ATPase) conformation is shown to contain very little beta-sheet structure under all conditions. Absolute content of alpha-helix and random coil or beta-turn could not be determined because of interference in the amide I and III regions. However, the Ca2+-ATPase in sarcoplasmic reticulum appears to undergo a conformational change at 15-18 degrees C which involves removal of a portion of the tryptophan residues from an aqueous environment and an increase in alpha-helical content. This conformation change coincides with a change in slope of Arrhenius plots of ATP hydrolysis activity. Increasing concentrations of Ca2+ and Mg2+ appear to slightly decrease the alpha-helical content of sarcoplasmic reticulum protein.     

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).     

Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering.

We examined the temperature- and pressure-induced unfolding and aggregation of beta-lactoglobulin (beta-Lg) and its genetic variants A and B up to temperatures of 90 degrees C in the pressure range from 1 bar to 10 kbar. To achieve information simultaneously on the secondary, tertiary, and quaternary structures, we have applied Synchrotron small-angle X-ray diffraction and Fourier transform infrared spectroscopy. Upon heating a beta-Lg solution at pH 7.0, the radius of gyration Rg first decreases, indicating a partial dissociation of the dimer into the monomers, the secondary structures remaining essentially unchanged. Above 50 degrees C, the infrared spectroscopy data reveal a decrease in intramolecular beta-sheet and alpha-helical structures, whereas the contribution of disordered structures increases. Within the temperature range from 50 to 60 degrees C, the appearance of the pair distance distribution function is not altered significantly, whereas the amount of defined secondary structures declines approximately by 10%. Above 60 degrees C the aggregation process of 1% beta-Lg solutions is clearly detectable by the increase in Rg and intermolecular beta-sheet content. The irreversible aggregation is due to intermolecular S-H/S-S interchange reactions and hydrophobic interactions. Upon pressurization at room temperature, the equilibrium between monomers and dimers is also shifted and dissociation of dimers is induced. At pressures of approximately 1300 bar, the amount of beta-sheet and alpha-helical structures decreases and the content of disordered structures increases, indicating the beginning unfolding of the protein which enables aggregation. Contrary to the thermal denaturation process, intermolecular beta-sheet formation is of less importance in pressure-induced protein aggregation and gelation. The spatial extent of the resulting protein clusters is time- and concentration-dependent. The aggregation of a 1% (w/w) solution of A, B, and the mixture AB results in the formation of at least octameric units as can be deduced from the radius of gyration of about 36 A. No differences in the pressure stability of the different genetic variants of beta-Lg are detectable in our FT-IR and SAXS experiments. Even application of higher pressures (up to 10 kbar) does not result in complete unfolding of all beta-Lg variants.     

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.     

Conformation and stability of Sendai virus fusion protein

The conformation and stability of Sendai virus fusion (F) protein were studied by circular dichroism spectroscopy, and the protein predictive models of Chou and Fasman and Robson and Suzuki were used to elucidate the secondary structure of Sendai virus F protein. The F protein conformation is predicted to contain 33% alpha-helix, 53% beta-sheet and 15% beta-turn by the Chou and Fasman model, and 30% alpha-helix, 55% beta-sheet, 9% beta-turn and 7% random coil by the Robson and Suzuki model. C.d. studies of F protein purified in the presence of the non-ionic detergent, n-octylglucoside, indicated the presence of 49% alpha-helix and 31% beta-sheet at pH 7.0, 54% alpha-helix and 28% beta-sheet at pH 9.0 and 50% alpha-helix and 23% beta-sheet at pH 5.4. A small change in conformation of the protein occurred when the pH was titrated from 7.0 to 5.4 and from 7.0 to 9.0 and a more pronounced conformational change occurred when the pH was changed from 9.0 to 5.4. The F protein in 0.2% n-octylglucoside was resistant to denaturation by 4 M guanidine hydrochloride, the reducing agent 20 mM mercaptoethanol, and to increases in temperature from 5 to 80 degrees C. Monoclonal anti-F protein antibody showed an increased binding to whole virus when the pH was changed from 7.0 to 9.0. The antibody binding was decreased when the pH was shifted from 9.0 to 5.4 Maximum haemolytic activity was observed with virus that was preincubated at pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a common intermediate of pea lectin in the folding pathway induced by TFE and HFIP

When pea lectin was exposed to a low pH range, it was found that the secondary structure of the lectin resisted conformational changes to a large extent up to pH 2.4 and below this pH, a sharp transition was observed which could be due to the presence of 27 acidic amino acid residues present in the protein. The effects of 1,1,1,3,3,3 hexafluoro-isopropanol (HFIP) and 2,2,2-Trifluoroethanol (TFE) on the conformation of pea lectin at pH 2.4 were studied using circular dichroism and fluorescence spectroscopy. Analysis varying the TFE concentration showed that up to 80% TFE (v/v) protein retained the residual beta-structure accompanied by a loss in tertiary structure. A similar conformation is presumed to exist at 4% HFIP (v/v), with an increase in HFIP concentration structural rearrangements occurred and a transition from beta-structure to alpha-helical structure started from 12% HFIP which completed at 30% HFIP. Our studies show the occurrence of a common intermediate in the folding pathway of pea lectin induced by two different fluoroalcohols, which differ in their mode of action to stabilize the secondary structure of a given protein. While TFE was not found to induce any alpha-helical structure, HFIP caused the transition of pea lectin, which is predominantly a beta-sheet protein, to a structure rich in alpha-helical contacts. Thus, our results also point out the possibility of a non-hierarchical model of protein folding in lectins.     

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.     

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.